Versatile Cell Culture Protocols for Diverse Cell Lines

HEK293A, HEK293P, HEK293T, H2.35, NIH3T3, C2C12, and HeLa cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) (Invitrogen) and 50 µg/ml penicillin/streptomycin in a humidified incubator with 5% CO₂. MCF10A cells were cultured in DMEM/F12 supplemented with 5% horse serum, 20 ng/ml EGF, 0.5 µg/ml hydrocortisone, 10 µg/ml insulin, 100 ng/ml cholera toxin, and 50 µg/ml penicillin/streptomycin. HepG2 cells were cultured in RPMI1640 supplemented with 10% FBS and 50 µg/ml penicillin/streptomycin. For glucose starvation, cells were washed twice with PBS and then incubated in DMEM without glucose and pyruvate (0 g/L glucose, 0 mM pyruvate; Invitrogen) supplemented with 10% FBS, Dialyzed (Invitrogen). Lats1 KO MEF cells, provided by Dr. Dae-SiK Lim (Korea Advanced Institute of Science and Technology), were maintained in DMEM with 10% FBS ^{39 (link)}. AMPKα WT and AMPKα DKO MEFs, a gift from Dr. Benoit Viollet, were cultured in DMEM medium with 10% FBS ^{40 (link)}. PolyJet™ DNA In Vitro Tranfection reagent (SignaGen Lab.) was used for transfection. siRNAs were synthesized by Dharmacon and delivered into cells using RNAiMAX (Invitrogen) according to manufacturer’s instructions.